The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

Marcello Ceccarelli; Luca Galluzzi; Aurora Diotallevi; Francesca Andreoni; Hailie Fowler; Christine Petersen; Fabrizio Vitale; Mauro Magnani

doi:10.1186/s13071-017-2181-x

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The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

Journal article

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The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

Marcello Ceccarelli, Luca Galluzzi, Aurora Diotallevi, Francesca Andreoni, Hailie Fowler, Christine Petersen, Fabrizio Vitale and Mauro Magnani

Parasites & vectors, Vol.10(1), pp.239-239

05/16/2017

DOI: 10.1186/s13071-017-2181-x

PMCID: PMC5434583

PMID: 28511704

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Abstract

Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the C values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of C values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Leishmaniasis - diagnosis

DNA, Kinetoplast - genetics

Species Specificity

Organic Chemicals

Leishmania - classification

DNA Primers

Sequence Analysis, DNA

Leishmania braziliensis - genetics

Animals

Leishmaniasis - veterinary

Sensitivity and Specificity

Dogs

Leishmaniasis - parasitology

Dog Diseases - diagnosis

Leishmania infantum - genetics

Real-Time Polymerase Chain Reaction

Transition Temperature

Dog Diseases - parasitology

Leishmania - genetics

Details

Title: Subtitle: The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis
Creators: Marcello Ceccarelli - Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy
Luca Galluzzi - Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy. luca.galluzzi@uniurb.it
Aurora Diotallevi - Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy
Francesca Andreoni - Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy
Hailie Fowler - Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, USA
Christine Petersen - Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, USA
Fabrizio Vitale - Istituto Zooprofilattico Sperimentale of Sicily "A Mirri", Palermo, PA, Italy
Mauro Magnani - Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, PU, Italy
Resource Type: Journal article
Publication Details: Parasites & vectors, Vol.10(1), pp.239-239
DOI: 10.1186/s13071-017-2181-x
PMID: 28511704
PMCID: PMC5434583
NLM abbreviation: Parasit Vectors
ISSN: 1756-3305
eISSN: 1756-3305
Publisher: England
Grant note: DOI: 10.13039/501100003196, name: Ministero della Salute, award: IZS SI /13 RC
Language: English
Date published: 05/16/2017
Academic Unit: Epidemiology; Internal Medicine
Record Identifier: 9983995047102771

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